The volume-regulated anion channel as a regulator of secretory activity in the intestinal epithelium

容量调节阴离子通道作为肠上皮分泌活性的调节剂

• 批准号: 10464156
• 负责人: John Thomas Gebert
• 金额: $ 4.71万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10464156
• 关键词: Ablation; Address; Adenosine Diphosphate; Age; Anions; Antidiarrheals; Biological Assay; Biosensor; CRISPR/Cas technology; Calcium; Calcium Oscillations; Cells; Child; Data; Diarrhea; Discipline; Dysentery; Enterocytes; Epithelial; Epithelial Cells; Equilibrium; Event; Fluids and Secretions; Frequencies; Gastrointestinal tract structure; Genes; Genetic; Health; Homeostasis; Human; Image; Infection; Intestines; Ions; Knock-out; Knowledge; Lead; Life; Liquid substance; Mediating; Mission; Morbidity - disease rate; Mus; Nonstructural Protein; Organoids; Paracrine Communication; Pathogenesis; Pathologic Processes; Pathway interactions; Pharmacology; Phenotype; Physiologic pulse; Play; Protein Kinase; Proteins; Recombinants; Regulation; Research; Rho-associated kinase; Role; Rotavirus; Rotavirus Infections; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Swelling; Testing; Therapeutic; United States National Institutes of Health; Variant; Viral; Work; base; burden of illness; clinically relevant; imaging genetics; in vivo; insight; intercellular communication; intestinal epithelium; knock-down; mortality; mouse model; new therapeutic target; novel; optogenetics; pathogenic virus; purinoceptor P2Y1; rho; small hairpin RNA; stressor; targeted treatment; tool

PROJECT SUMMARY The intestinal epithelium relies on extensive cell-cell signaling to maintain a tight balance between absorptive and secretory activity, but the intricacies of this signaling remain uncharacterized. We recently discovered that cells infected with rotavirus, the most common cause of life-threatening diarrhea worldwide, release hundreds of pulses of adenosine diphosphate (ADP) causing intercellular calcium waves that dysregulate uninfected cells. Blocking the ADP receptor P2Y1 significantly reduces diarrhea severity in rotavirus-infected mice, suggesting that this signaling is integral in regulating secretory activity. While the mechanism through which rotavirus triggers the release of ADP has not been confirmed, our preliminary data show it relies on activation of the volume- regulated anion channel (VRAC). VRAC is increasingly recognized as a conduit for paracrine signals associated with a variety of intracellular stressors, but it has yet to be studied within the epithelium. Further, the mechanisms regulating its activation in any context remain poorly understood. Therefore, the objective of this proposal is to use rotavirus as a tool to study the role and activation of VRAC in the intestinal epithelium. Given that rotavirus-induced intercellular calcium waves are inhibited by knock down of rotavirus non-structural protein 4 (NSP4), a Ca2+-conducting channel, my central hypothesis is that NSP4-induced elevations of cytosolic Ca2+ activate host pathways that trigger ADP release through VRAC, amplifying secretory activity throughout the epithelium. I will test this hypothesis by using long-term live calcium imaging in conjunction with novel fluorescent Rho biosensors and CRISPR-Cas9 based gene editing to [Aim 1] determine the intracellular pathway mediating VRAC activation in RV-infected cells. Given ADP release is reduced upon pharmacological inhibition of Rho kinase or knockdown of the calcium-augmenting rotavirus protein NSP4, we expect both calcium and Rho are involved in VRAC activation. Using both human intestinal enteroids and the mouse model of rotavirus diarrhea, I will [Aim 2] determine the effect of VRAC activation on secretory activity in the intestinal epithelium. This work will identify novel therapeutic targets for the treatment of secretory diarrhea, a leading cause of mortality among children worldwide. Furthermore, our findings will address gaps in knowledge surrounding VRAC activation and its role in paracrine signaling. Given that VRAC has been implicated in an increasing variety of pathological processes, using this clinically relevant approach will yield information translatable to both human health and other scientific disciplines.

项目摘要 肠上皮细胞依赖于广泛的细胞间信号传导来维持吸收和分泌之间的紧密平衡。 和分泌活动，但这种复杂的信号仍然没有特征。我们最近发现 轮状病毒是全球威胁生命的腹泻的最常见原因，感染轮状病毒的细胞会释放数百种 二磷酸腺苷（ADP）脉冲导致未感染细胞的细胞间钙波失调。 阻断ADP受体P2 Y1可显著降低轮状病毒感染小鼠的腹泻严重程度，这表明 这种信号在调节分泌活动中是不可或缺的。虽然轮状病毒触发 ADP的释放尚未得到证实，我们的初步数据显示它依赖于体积的激活- 调节阴离子通道（VRAC）。VRAC越来越被认为是旁分泌信号的通道 与多种细胞内应激源相关，但尚未在上皮内进行研究。此夕h 在任何情况下调节其激活的机制仍然知之甚少。因此，这一目标 建议使用轮状病毒作为研究VRAC在肠上皮中的作用和激活的工具。给定 轮状病毒诱导的细胞间钙波被轮状病毒非结构蛋白的敲低所抑制 4（NSP 4），一种Ca 2+传导通道，我的中心假设是NSP 4诱导的细胞质中 Ca 2+激活宿主途径，通过VRAC触发ADP释放，放大分泌活性 遍布整个上皮。我将通过使用长期活体钙成像结合 使用新型荧光Rho生物传感器和基于CRISPR-Cas9的基因编辑来[目的1]确定 在RV感染的细胞中，细胞内途径介导VRAC活化。鉴于ADP释放量减少， Rho激酶的药理学抑制或钙增加轮状病毒蛋白NSP 4的敲低，我们 预期钙和Rho均参与VRAC激活。使用人类肠类和 在轮状病毒腹泻小鼠模型中，我将[目的2]确定VRAC激活对 肠上皮这项工作将确定治疗分泌性腹泻的新的治疗靶点， 是全球儿童死亡的主要原因。此外，我们的研究结果将解决知识差距， 周围VRAC激活及其在旁分泌信号传导中的作用。鉴于VRAC参与了 越来越多的病理过程，使用这种临床相关的方法将产生信息 可用于人类健康和其他科学学科。